This invention relates to nucleic acid and amino acid sequences of a novel human mitochondrial chaperone protein and to the use of these sequences in the diagnosis, prevention, and treatment of cancer and disorders associated with fungal and parasitic infections.
The majority of mitochondrial proteins are encoded by nuclear genes, are synthesized on cytosolic ribosomes, and are imported into the mitochondria. Nuclear-encoded proteins which are destined for the mitochondrial matrix typically contain positively-charged amino terminal signal sequences. Import of these preproteins from the cytoplasm requires a multisubunit protein complex in the outer membrane known as the translocase of outer mitochondrial membrane (TOM; previously designated MOM; Pfanner, N. et al. (1996) Trends Biochem. Sci. 21:51-52) and at least three inner membrane proteins which comprise the translocase of inner Mitochondrial membrane (TIM; previously designated MIM; Pfanner et al, supra). An inside-negative membrane potential across the inner mitochondrial membrane is also required for preprotein import. Preproteins are recognized by surface receptor components of the TOM complex and are translocated through a proteinaceous pore formed by other TOM components. Proteins targeted to the matrix are then recognized and transported by the import machinery of the TIM complex.
As the unfolded preprotein emerges into mitochondrial matrix, it binds to a matrix protein termed mt-Hsp70 (also known as Ssc1p in yeast) which facilitates the completion of preprotein import. The mt-Hsp70 protein is a member of a ubiquitous family of heat shock inducible molecular chaperone molecules homologous to the E. coli DnaK protein. An additional molecular chaperone essential for preprotein import is the matrix protein mt-GrpE (also known in yeast as Mge1p or Yge1p), a 24 kd homolog of the prokaryotic heat shock chaperone protein GrpE. The binding of preproteins to mt-Hsp70 is dependent upon a cycle of ATP binding and hydrolysis which is regulated in part by mt-GrpE, possibly by direct interaction with mt-Hsp70 (Pfanner, N. et al. (1995) Curr. Biol. 5:132-135). In yeast, both mt-Hsp70 and mt-GrpE are essential for viability (Pfanner et al., (supra); Laloraya, S. et al. (1994) Proc. Natl. Acad. Sci. USA 91:6481-6485).
Members of the Hsp70 family are highly conserved among bacterial to mammalian species. Hsp70 proteins maintain at least 50% positional identity across the evolutionary spectrum (Webster, T. J. et al. (1994) DNA Cell Biol 13:1213-1220). The GrpE family is less evolutionarily conserved; a cDNA encoding mt-GrpE from rat shows only 20 to 30% positional identity with its bacterial and fungal counterparts (Naylor, D. J. et al. (1996) FEBS Letts. 396:181-188). Rat mt-GrpE is translated in the cytosol as a 217 amino acid preprotein. The highly basic 27 amino acid N-terminal mitochondrial targeting sequence is proteolytically removed upon import into the mitochondrial matrix (Naylor et al., supra).
Fungal infections are major health problems, especially among immunocompromised individuals. Patients are immunosuppressed to prevent the rejection of transplants and to treat neoplastic and inflammatory diseases. In addition, some infections, most notably that caused by human immunodeficiency virus (HIV), immunocompromise the host. Infectious agents that coexist peacefully with immunocompetent hosts wreak havoc in those who lack a complete immune system. Pulmonary infections by fungi such as Histoplasma sp. and Coccidioides immitis may be fatal in immunocompromised individuals, young children, or elderly patients. Patients with diabetes mellitus or hematologic malignancy, or those receiving broad-spectrum antibiotics or high doses of adrenal corticosteroids, are especially susceptible to tissue invasion by Candida. Aspergillus is another widespread fungus which does not commonly cause disease except in immunocompromised patients.
Protozoan parasites cause widespread and debilitating diseases in humans and domestic livestock throughout the tropics. Examples of these diseases include malaria (caused by Plasmodium falciparum), African sleeping sickness and the cattle disease nagana (caused by Trypanosoma brucei), Chagas"" disease (caused by Trypanosoma cruzi), and kala azar, espundia, and Oriental sore (caused by Leishmania sp.). There are no vaccines against these diseases, and most of the available drug treatments are toxic and/or ineffective. Recently, drug resistant Plasmodium has placed malaria back into this category. The World Health Organization has identified the development of new and safer treatments for these diseases as a major priority.
Discovery of a novel human mitochondrial chaperone protein and the polynucleotides which encode it satisfies a need in the art by providing new compositions useful in diagnosing and treating cancer. Knowledge and expression of sequences encoding the novel human mitochondrial chaperone protein is also useful for developing therapeutic agents to prevent or treat diseases associated with fungal and parasitic infections.
The present invention features a novel human mitochondrial chaperone protein hereinafter designated Hmt-GrpE and characterized as having similarity to mt-GrpE protein from rat, Droel from fruit fly, and Mge1p from baker""s yeast.
Accordingly, the invention features a substantially purified Hmt-GrpE having the amino acid sequence shown in SEQ ID NO:1.
One aspect of the invention features isolated and substantially purified polynucleotides that encode Hmt-GrpE. In a particular aspect, the polynucleotide is the nucleotide sequence of SEQ ID NO:2.
The invention also relates to a polynucleotide sequence comprising the complement of SEQ ID NO:2 or variants thereof. In addition, the invention features polynucleotide sequences which hybridize under stringent conditions to SEQ ID NO:2.
The invention additionally features nucleic acid sequences encoding polypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments, portions or antisense molecules thereof, and expression vectors and host cells comprising polynucleotides that encode Hmt-GrpE. The present invention also features antibodies which bind specifically to Hmt-GrpE. The invention also features antagonists of Hmt-GrpE and the use thereof in treating cancer. The invention also features methods for detecting a polynucleotide which encodes Hmt-GrpE and for the use of Hmt-GrpE in identifying antifungal and antiparasitic therapeutics.